Project Summary ? Project 1 Tumor progression is the product genetic mutations that affect gene function and epigenetic alterations that affect patterns of gene expression and cellular states. Together with the effects of the tumor microenvironment, these changes produce the phenotypes that underlie the biology of cancer. From extensive genomic studies performed over the past decade the genetic landscape of cancer progression is beginning to emerge, but much less is know about how tumors evolve epigenetically. Project 1 is focused on the investigation of tumor evolution through the lens of changes in epigenetic states and gene expression programs. It is anticipated that an improved understanding of these aspects of tumor development will yield new strategies to intervene in this process as well as to treat established tumors. Three Specific Aims will be pursued addressing these questions in the context of well-established genetically-engineered mouse models of human lung adenocarcinoma (LUAD) based on mutations of K-ras (?K?) or the combination of K-ras and p53 (?KP?). Of note, K-ras mutations occur in approximately 30% of human LUAD and targeted therapies do not exist for this subtype of the disease. Preliminary data in support of the Project 1 demonstrates that as tumors progress in the KP model they develop subpopulations of cells that have cancer stem-cell like properties and show markers of response to the Wnt signaling pathway and other cells that markers of Wnt-producing cells that express the enzyme Porcupine (Porcn). It is hypothesized that these Porcn+ cells act as niche cells for the cancer stem-like cells. In Aim 1, a combination of genetic marking, molecular profiling and cell ablation studies will be used to characterized and functionally test these putative niche cells for their importance in tumor development. These studies may identify new targets for the treatment of human lung cancer. Using single cell mRNA sequencing (SCmRNAseq), performed in collaboration with the laboratory of Aviv Regev, the Jacks laboratory has profiled individual cancer cells from K and KP tumors. These data reveal extensive intratumoral and intertumoral transcriptional heterogeneity. Aim 2 of Project 1 will complete the characterization of transcriptional heterogeneity as a function of tumor genotype and tumor progression as well as explore the epigenetic mediators of these effects. Finally, the transcriptional programs of cancer cells are expected to be affected by immune infiltration and anti-tumor immune responses, potentially in a heterogeneous fashion across tumors. These questions will be explored in Aim 3 of Project 1 using SCmRNAseq-based profiling and functional studies of ?immunogenic and ?nonimmunogenic? tumors. The identification and functional validation of pathways that contribute to immune suppression in this model could reveal new targets of therapy for human lung cancer and other cancer types. In all of the Aims of Project 1, a series of innovative genetic technologies (including lineage tracing, cell marking, cell ablation, and CRISPR/Cas9-mediated genome editing and gene activation) will be used to functionally characterization of genes and pathways identified through profiling.